In the biological or medical field, a protein of interest to be produced is obtained by inserting the gene of the protein into an expression vector, transfecting the expression vector into a cell line producing the protein, culturing the resulting cell line in a large amount, and isolating and purifying the cultured cell line using a suitable method.
Examples of methods which are industrially used for this purpose include methods that use CHO (Chinese hamster ovary) dhfr (dihydrofolate reductase) (−), CHO K1, BHK (Baby Hamster Kidney), NSO, SP2/0 and human cell lines (Ogata, et al., Appl. Microbiol. Biotechnol., 1993, 38(4), 520-525; Kratje, et al., Biotechnol. Prog., 1994, 10(4), 410-20; Peakman, et al., Hum. Antibodies Hybridomas, 1994, 5(1-2), 65-74).
In order to produce a stable mammalian cell line which expresses a heterologous gene of interest, the heterologous gene is generally introduced into the desired cell line together with a selectable marker gene (e.g. neomycin phosphotransferase) by transfection. The heterologous gene and the selectable marker gene can be expressed either together by a single vector or by separate vectors which are co-transfected. Two to three days after transfection, the cells are transferred into a medium containing a selective agent, for example, G418 in case of using neomycin phosphotransferase-gene as a selectable marker gene, and cultured for several weeks under these selective conditions. The emergent resistance cells can then be isolated and investigated for expression of the desired gene product. As a result of the random and non-directed integration into the host cell genome, populations of cells are obtained which have completely different rates of expression of the heterologous gene. These may also include non-expressing cells in which the selectable marker is expressed but not the gene of interest. In order to identify cell clones which have a very high expression of the heterologous gene of interest, it is therefore necessary to examine and test a large number of clones, which is time-consuming, labor-intensive and expensive.
Gene amplification is a common technique in animal cell cultures for use in the production of recombinant proteins. The gene amplification significantly improves the relatively low productivity of numerous mammalian cell lines. One amplification technique widely used in the art is a dihydrofolate reductase (dhfr)-based gene amplification system which is very often used in dhfr-deficient CHO cells.
The reasons why the dhfr-deficient CHO cell line is industrially preferred are as follows: (1) posttranslational modification of protein (glycosylation or phosphorylation) is more similar to that of human cells than other cells; (2) not only adhesion culture, but also suspension culture are possible; (3) these cells can be cultured at relatively high concentrations in a serum-free medium compared to other cells; (4) the productivity of the protein of interest, which is significantly low compared to that of microorganisms, can be increased using a dhfr/MTX (methotrexate) gene amplification system; and (5) these cells have been proven safe and can thus be easily approved by relevant authorities such as the FDA. For the above-described reasons, the dhfr-deficient CHO cell line has been widely used for industrial purposes to construct a recombinant cell line producing a protein of interest.
However, even when the conventional dhfr/MTX gene amplification system is used that can significantly increase the production of the protein of interest via introduction of the gene encoding the protein of interest into dhfr-deficient CHO cells and then treatment of the cells with stepwise increasing concentrations of MTX, selecting cell populations having high productivity is labor-intensive, time and cost-consuming, because a process of treating the cells with increasing concentrations of MTX for a long time is required and the number of cell populations to be screened in this process is 500-4,000 or more. Accordingly, efforts to simplify this selection have been attempted in various ways.
For example, researchers in the art have made efforts to simplify the selection step using 1) a method of reducing the expression of a selectable marker gene by modifying the vicinity of the start codon (Reff, U.S. Pat. No. 5,733,779, 1998); 2) a method of impairing the function of a selectable marker gene by mutating the gene (Nivea et al., Gene 108, 193-200, 1991; Sauter and Enenkel, Biotechnol. Bioeng. 89, 530-538, 2005); 3) a method of modifying the start codon itself and linking the modified start codon with a gene to be expressed, thereby increasing the expression level of the gene (van Blokland et al., J of Biotechnology 128, 237-245, 2007); and 4) a method of linking a selectable marker gene to an intron and expressing the gene (Lucas et al., Nucleic Acids Res. 24, 1774-1779, 1996).